The cardiac homeobox gene Csx/Nkx2.5 lies genetically upstream of multiple genes essential for heart development

Csx/Nkx2.5 is a vertebrate homeobox gene with a sequence homology to the Drosophila tinman, which is required for the dorsal mesoderm specification. Recently, heterozygous mutations of this gene were found to cause human congenital heart disease (Schott, J.-J., Benson, D. W., Basson, C. T., Pease, W., Silberbach, G. M., Moak, J. P., Maron, B. J., Seidman, C. E. and Seidman, J. G. (1998) Science 281, 108–111). To investigate the functions of Csx/Nkx2.5 in cardiac and extracardiac development in the vertebrate, we have generated and analyzed mutant mice completely null for Csx/Nkx2.5. Homozygous null embryos showed arrest of cardiac development after looping and poor development of blood vessels. Moreover, there were severe defects in vascular formation and hematopoiesis in the mutant yolk sac. Interestingly, TUNEL staining and PCNA staining showed neither enhanced apoptosis nor reduced cell proliferation in the mutant myocardium. In situ hybridization studies demonstrated that, among 20 candidate genes examined, expression of ANF, BNP, MLC2V, N-myc, MEF2C, HAND1 and Msx2 was disturbed in the mutant heart. Moreover, in the heart of adult chimeric mice generated from Csx/Nkx2.5 null ES cells, there were almost no ES cell-derived cardiac myocytes, while there were substantial contributions of Csx /Nkx2.5-deficient cells in other organs. Whole-mount β-gal staining of chimeric embryos showed that more than 20% contribution of Csx/Nkx2. 5-deficient cells in the heart arrested cardiac development. These results indicate that (1) the complete null mutation of Csx/Nkx2.5 did not abolish initial heart looping, (2) there was no enhanced apoptosis or defective cell cycle entry in Csx/Nkx2.5 null cardiac myocytes, (3) Csx/Nkx2.5 regulates expression of several essential transcription factors in the developing heart, (4) Csx/Nkx2.5 is required for later differentiation of cardiac myocytes, (5) Csx/Nkx2. 5 null cells exert dominant interfering effects on cardiac development, and (6) there were severe defects in yolk sac angiogenesis and hematopoiesis in the Csx/Nkx2.5 null embryos.

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The Expanding Role for Retinoid Signaling in Heart Development

The Scientific World JOURNAL ◽

10.1100/tsw.2008.39 ◽

2008 ◽

Vol 8 ◽

pp. 194-211 ◽

Cited By ~ 20

Author(s):

Loretta L. Hoover ◽

Elizabeth G. Burton ◽

Bonnie A. Brooks ◽

Steven W. Kubalak

Keyword(s):

Vitamin A ◽

Heart Development ◽

Cardiac Development ◽

Tube Formation ◽

Conditional Knockout ◽

Heart Tube ◽

Developmental Defects ◽

Retinoid Signaling ◽

Developing Heart ◽

Cardiac Lineage

The importance of retinoid signaling during cardiac development has long been appreciated, but recently has become a rapidly expanding field of research. Experiments performed over 50 years ago showed that too much or too little maternal intake of vitamin A proved detrimental for embryos, resulting in a cadre of predictable cardiac developmental defects. Germline and conditional knockout mice have revealed which molecular players in the vitamin A signaling cascade are potentially responsible for regulating specific developmental events, and many of these molecules have been temporally and spatially characterized. It is evident that intact and controlled retinoid signaling is necessary for each stage of cardiac development to proceed normally, including cardiac lineage determination, heart tube formation, looping, epicardium formation, ventricular maturation, chamber and outflow tract septation, and coronary arteriogenesis. This review summarizes many of the significant milestones in this field and particular attention is given to recently uncovered cross-talk between retinoid signaling and other developmentally significant pathways. It is our hope that this review of the role of retinoid signaling during formation, remodeling, and maturation of the developing heart will serve as a tool for future discoveries.

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Vinculin knockout results in heart and brain defects during embryonic development

Development ◽

10.1242/dev.125.2.327 ◽

1998 ◽

Vol 125 (2) ◽

pp. 327-337 ◽

Cited By ~ 22

Author(s):

W. Xu ◽

H. Baribault ◽

E.D. Adamson

Keyword(s):

Embryonic Development ◽

Focal Adhesion ◽

Heart Development ◽

Cardiac Development ◽

Es Cells ◽

Embryonic Stem ◽

Spinal Nerve ◽

Cell Behaviors ◽

Migration Rates ◽

Targeting Vector

The vinculin gene codes for a cytoskeletal protein, found in focal adhesion plaques and in cell-cell adherens junctions. Vinculin was inactivated by homologous recombination using a targeting vector in embryonic stem (ES) cells. The heterozygous ES cells were introduced into mice by established procedures to produce heterozygous animals that were normal and fertile. No homozygous vinculin−/− embryos were born and analyses during the gestational period showed that the vinculin null embryos were small and abnormal from day E8 but some survived until E10. The most prominent defect was lack of midline fusion of the rostral neural tube, producing a cranial bilobular appearance and attenuation of cranial and spinal nerve development. Heart development was curtailed at E9.5, with severely reduced and akinetic myocardial and endocardial structures. Mutant embryos were 30–40% smaller, somites and limbs were retarded and ectodermal tissues were sparse and fragile. Fibroblasts (MEF) isolated from mutant embryos were shown to have reduced adhesion to fibronectin, vitronectin, laminin and collagen compared to wild-type levels. In addition, migration rates over these substrata were two-fold higher and the level of focal adhesion kinase (FAK) activity was three-fold higher. We conclude that vinculin is necessary for normal embryonic development, probably because of its role in the regulation of cell adhesion and locomotion, cell behaviors essential for normal embryonic morphogenesis, although specific roles in neural and cardiac development cannot be ruled out.

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Abstract 1461: Wnt2 Plays a Key Role in Cardiac Development Via a Non-Canonical Pathway

Circulation ◽

10.1161/circ.118.suppl_18.s_330 ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Takeshi Onizuka ◽

Shinsuke Yuasa ◽

Kenichiro Shimoji ◽

Keiichi Fukuda ◽

Satoshi Ogawa

Keyword(s):

Heart Development ◽

Cardiac Development ◽

Early Stage ◽

Es Cells ◽

Embryonic Stem ◽

Canonical Pathway ◽

Embryonic Cells ◽

Potent Effect ◽

Canonical Wnt ◽

Promising Source

Embryonic stem (ES) cells are a promising source of cardiomyocytes, but their clinical application has been hindered by the lack of selective differentiation methods. Although several signals are involved in heart development, the precise signals that mediate cardiomyocyte differentiation remain undetermined. Wnt family has a potent effect on the various organ development. To address this issue, we investigated the expression of wnt genes in the embryonic heart. Then we applied these findings to establish an efficient protocol to induce cardiomyocytes in vitro . (1) We analyzed TOP-EGFP mice to clarify whether canonical wnt signal pathway is important in the developing heart. TOP-EGFP mice are transgenic mice in which the EGFP gene is located under the β-catenin binding site so that the EGFP protein expresses when the canonical pathway is activated. They did not reveal any GFP in early stage heart, indicating that the canonical pathway is not involved. (2) Expression of non-canonical wnt was screened. Whole mount in situ hybridization of wnt2 and nkx2.5 (positive control) was performed at mouse embryo. Wnt2 was strongly expressed in the the heart-forming area in stages from E7.5 to E9.0. (3) We applied this embryonic wnt2 expression pattern to ES cell differentiation. Using siRNA we knocked-down wnt2 protein in various phases, which inhibited formation of beating EB, and decreased cardiac muscle genes only during the primitive stage (day 2– 4). Also adding wnt2 protein to embryonic cells in the appropriate phase led to a marked induction of cardiac specific genes. But wnt2 did not affect the mesodermal gene expression, Brachyury T and Mesp1, suggesting that Wnt2 does not affect the primitive development of the mesodermal progenitor cells. However, Wnt2 critically promotes cardiac specification after mesodermal induction and increases the eventual cardiac musculature. Wnt2 was strongly expressed in the heart-forming region. We applied this finding to develop an effective protocol for obtaining cardiomyocytes from mouse ES cells by adding wnt2 protein and also to inhibit generation of cardiomyocytes by inhibition of wnt2 signaling. We concluded that wnt2 plays a key role in cardiac development via a non-canonical pathway.

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Cardiac Defects Associated with the Absence of the Platelet-derived Growth Factor α Receptor in the Patch Mouse

Microscopy and Microanalysis ◽

10.1007/s100050010061 ◽

2001 ◽

Vol 7 (1) ◽

pp. 56-65

Author(s):

Robert L. Price ◽

Thomas E. Thielen ◽

Thomas K. Borg ◽

Louis Terracio

Keyword(s):

Growth Factor ◽

Cardiac Myocytes ◽

Heart Development ◽

Cardiac Development ◽

Outflow Tract ◽

Cytoskeletal Proteins ◽

Platelet Derived Growth Factor ◽

Mouse Heart ◽

Littermate Control ◽

Naturally Occurring

AbstractIn this report, we describe the distribution of the platelet-derived growth factor receptor α (PDGFRα) by immunolocalization in the embryonic day 10.5 mouse heart and defects in heart development associated with the absence of this receptor in the Patch mouse. The Patch mouse is a naturally occurring mutant that has been accepted as a model for determining the role of the PDGFRα in early cardiac development. Even though other genetic defects exist in this naturally occurring mutant, most defects associated with cardiac development are believed to be a result of the absence of this receptor. Gross morphological defects including improper septation of the outflow tract, dysmorphic shape of the heart, and lack of trabecular development are similar to those that have been previously described. Many of these defects have been attributed to the failure of a subset of non-neuronal neural crest cells to properly migrate into the region of the developing outflow tract. In these studies, we have also used confocal scanning laser and transmission electron microscopy to describe and compare the organization and differentiation of the cytoskeletal proteins actin and myosin in littermate control and Patch mouse hearts. Cytoskeletal organization of the cardiac myocytes in Patch mouse hearts has not previously been described. In most cardiac myocytes of Patch mice, actin was found only on the periphery of the cells, and the organization of actin, myosin, and precursor Z-band material into distinct myofibrils was greatly reduced.

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Construction and Establishment of a New Environmental Chamber to Study Real-Time Cardiac Development

Microscopy and Microanalysis ◽

10.1017/s1431927607070390 ◽

2007 ◽

Vol 13 (3) ◽

pp. 204-210 ◽

Cited By ~ 6

Author(s):

Gülay Orhan ◽

Stephan Baron ◽

Kambiz Norozi ◽

Jörg Männer ◽

Oliver Hornung ◽

...

Keyword(s):

Real Time ◽

Heart Development ◽

Limb Development ◽

Cardiac Development ◽

Time Window ◽

Time Lapse ◽

Cardiac Looping ◽

Developing Heart ◽

Stable Conditions ◽

Video Microscope

Heart development, especially the critical phase of cardiac looping, is a complex and intricate process that has not yet been visualized “live” over long periods of time. We have constructed and established a new environmental incubator chamber that provides stable conditions for embryonic development with regard to temperature, humidity, and oxygen levels. We have integrated a video microscope in the chamber to visualize the developing heart in real time and present the first “live” recordings of a chick embryo in shell-less culture acquired over a period of 2 days. The time-lapse images we show depict a significant time window that covers the most critical and typical morphogenetic events during normal cardiac looping. Our system is of interest to researchers in the field of embryogenesis, as it can be adapted to a variety of animal models for organogenesis studies including heart and limb development.

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Myocardin Expression Is Regulated by Nkx2.5, and Its Function Is Required for Cardiomyogenesis

Molecular and Cellular Biology ◽

10.1128/mcb.23.24.9222-9232.2003 ◽

2003 ◽

Vol 23 (24) ◽

pp. 9222-9232 ◽

Cited By ~ 78

Author(s):

Tomomi Ueyama ◽

Hideko Kasahara ◽

Takahiro Ishiwata ◽

Qing Nie ◽

Seigo Izumo

Keyword(s):

Heart Development ◽

Transforming Growth Factor ◽

Homeobox Gene ◽

Transforming Growth Factor Β ◽

Subtractive Hybridization ◽

Bone Morphogenetic Protein 2 ◽

Null Mouse ◽

Teratocarcinoma Cell ◽

Mouse Cdna ◽

Developing Heart

ABSTRACT Nkx2.5 (also known as Csx) is an evolutionarily conserved cardiac transcription factor of the homeobox gene family. Nkx2.5 is required for early heart development, since Nkx2.5-null mice die before completion of cardiac looping. To identify genes regulated by Nkx2.5 in the developing heart, we performed subtractive hybridization by using RNA isolated from wild-type and Nkx2.5-null hearts at embryonic day 8.5. We isolated a mouse cDNA encoding myocardin A, which is an alternative spliced isoform of myocardin and the most abundant isoform in the heart from embryo to adult. The expression of myocardin A and myocardin was markedly downregulated in Nkx2.5-null mouse hearts. Transient-cotransfection analysis showed that Nkx2.5 transactivates the myocardin promoter. Inhibition of myocardin function in the teratocarcinoma cell line P19CL6 prevented differentiation into cardiac myocytes after dimethyl sulfoxide treatment. Myocardin A transactivated the promoter of the atrial natriuretic factor gene through the serum response element, which was augmented by bone morphogenetic protein 2 and transforming growth factor β-activated kinase 1. These results suggest that myocardin expression is regulated by Nkx2.5 and that its function is required for cardiomyogenesis.

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The Iroquois Homeobox Gene Irx2 Is Not Essential for Normal Development of the Heart and Midbrain-Hindbrain Boundary in Mice

Molecular and Cellular Biology ◽

10.1128/mcb.23.22.8216-8225.2003 ◽

2003 ◽

Vol 23 (22) ◽

pp. 8216-8225 ◽

Cited By ~ 36

Author(s):

Mélanie Lebel ◽

Pooja Agarwal ◽

Chi Wa Cheng ◽

M. Golam Kabir ◽

Toby Y. Chan ◽

...

Keyword(s):

Heart Development ◽

Homeobox Gene ◽

Mutant Mice ◽

Phenotypic Analysis ◽

Functional Roles ◽

Developing Heart ◽

Dynamic Expression ◽

Evolutionarily Conserved ◽

And Function ◽

Deficient Mice

ABSTRACT The Iroquois homeobox (Irx) genes have been implicated in the specification and patterning of several organs in Drosophila and several vertebrate species. Misexpression studies of chick, Xenopus, and zebra fish embryos have demonstrated that Irx genes are involved in the specification of the midbrain-hindbrain boundary. All six murine Irx genes are expressed in the developing heart, suggesting that they might possess distinct functions during heart development, and a role for Irx4 in normal heart development has been recently demonstrated by gene-targeting experiments. Here we describe the generation and phenotypic analysis of an Irx2-deficient mouse strain. By targeted insertion of a lacZ reporter gene into the Irx2 locus, we show that lacZ expression reproduces most of the endogenous Irx2 expression pattern. Despite the dynamic expression of Irx2 in the developing heart, nervous system, and other organs, Irx2-deficient mice are viable, are fertile, and appear to be normal. Although chick Irx2 has been implicated in the development of the midbrain-hindbrain region, we show that Irx2-deficient mice develop a normal midbrain-hindbrain boundary. Furthermore, Irx2-deficient mice have normal cardiac morphology and function. Functional compensation by other Irx genes might account for the absence of a phenotype in Irx2-deficient mice. Further studies of mutant mice of other Irx genes as well as compound mutant mice will be necessary to uncover the functional roles of these evolutionarily conserved transcriptional regulators in development and disease.

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The ECM as a driver of heart development and repair

Development ◽

10.1242/dev.191320 ◽

2021 ◽

Vol 148 (5) ◽

pp. dev191320

Author(s):

Christopher J. Derrick ◽

Emily S. Noël

Keyword(s):

Extracellular Matrix ◽

Heart Development ◽

Cardiac Development ◽

Cardiac Cells ◽

Heart Regeneration ◽

Heart Morphogenesis ◽

Developing Heart ◽

Spatiotemporal Changes ◽

And Function

ABSTRACTThe developing heart is formed of two tissue layers separated by an extracellular matrix (ECM) that provides chemical and physical signals to cardiac cells. While deposition of specific ECM components creates matrix diversity, the cardiac ECM is also dynamic, with modification and degradation playing important roles in ECM maturation and function. In this Review, we discuss the spatiotemporal changes in ECM composition during cardiac development that support distinct aspects of heart morphogenesis. We highlight conserved requirements for specific ECM components in human cardiac development, and discuss emerging evidence of a central role for the ECM in promoting heart regeneration.

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The chromosomal protein SMCHD1 regulates DNA methylation and the 2c-like state of embryonic stem cells by antagonizing TET proteins

Science Advances ◽

10.1126/sciadv.abb9149 ◽

2021 ◽

Vol 7 (4) ◽

pp. eabb9149

Author(s):

Zhijun Huang ◽

Jiyoung Yu ◽

Wei Cui ◽

Benjamin K. Johnson ◽

Kyunggon Kim ◽

...

Keyword(s):

Es Cells ◽

Embryonic Stem ◽

Homeobox Gene ◽

Dna Demethylation ◽

Chromosomal Protein ◽

Dna Hypomethylation ◽

Tet Proteins ◽

Target Sites ◽

Structural Maintenance Of Chromosomes ◽

Hinge Domain

5-Methylcytosine (5mC) oxidases, the ten-eleven translocation (TET) proteins, initiate DNA demethylation, but it is unclear how 5mC oxidation is regulated. We show that the protein SMCHD1 (structural maintenance of chromosomes flexible hinge domain containing 1) is found in complexes with TET proteins and negatively regulates TET activities. Removal of SMCHD1 from mouse embryonic stem (ES) cells induces DNA hypomethylation, preferentially at SMCHD1 target sites and accumulation of 5-hydroxymethylcytosine (5hmC), along with promoter demethylation and activation of the Dux double-homeobox gene. In the absence of SMCHD1, ES cells acquire a two-cell (2c) embryo–like state characterized by activation of an early embryonic transcriptome that is substantially imposed by Dux. Using Smchd1/Tet1/Tet2/Tet3 quadruple-knockout cells, we show that DNA demethylation, activation of Dux, and other genes upon SMCHD1 loss depend on TET proteins. These data identify SMCHD1 as an antagonist of the 2c-like state of ES cells and of TET-mediated DNA demethylation.

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Abnormal Cardiac Development in the Absence of Heart Glycogen

Molecular and Cellular Biology ◽

10.1128/mcb.24.16.7179-7187.2004 ◽

2004 ◽

Vol 24 (16) ◽

pp. 7179-7187 ◽

Cited By ~ 68

Author(s):

Bartholomew A. Pederson ◽

Hanying Chen ◽

Jill M. Schroeder ◽

Weinian Shou ◽

Anna A. DePaoli-Roach ◽

...

Keyword(s):

Cardiac Function ◽

Cardiac Muscle ◽

Heart Development ◽

Congenital Heart ◽

Cardiac Development ◽

Glycogen Synthase ◽

Heart Defects ◽

Mendelian Inheritance ◽

And Function ◽

Impaired Cardiac Function

ABSTRACT Glycogen serves as a repository of glucose in many mammalian tissues. Mice lacking this glucose reserve in muscle, heart, and several other tissues were generated by disruption of the GYS1 gene, which encodes an isoform of glycogen synthase. Crossing mice heterozygous for the GYS1 disruption resulted in a significant underrepresentation of GYS1-null mice in the offspring. Timed matings established that Mendelian inheritance was followed for up to 18.5 days postcoitum (dpc) and that ∼90% of GYS1-null animals died soon after birth due to impaired cardiac function. Defects in cardiac development began between 11.5 and 14.5 dpc. At 18.5 dpc, the hearts were significantly smaller, with reduced ventricular chamber size and enlarged atria. Consistent with impaired cardiac function, edema, pooling of blood, and hemorrhagic liver were seen. Glycogen synthase and glycogen were undetectable in cardiac muscle and skeletal muscle from the surviving null mice, and the hearts showed normal morphology and function. Congenital heart disease is one of the most common birth defects in humans, at up to 1 in 50 live births. The results provide the first direct evidence that the ability to synthesize glycogen in cardiac muscle is critical for normal heart development and hence that its impairment could be a significant contributor to congenital heart defects.

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